To predict drug response or toxicity, the pharmaceutical industry is increasingly performing smaller-scale validation studies of experimental drug compounds in novel 3D cell culture models intended to mimic more closely the structure, activity, and extracellular environment of tissues in vivo.
Following high-throughput, large-scale screening assays performed in 2D systems, these smaller, secondary screens in 3D microtissue models are more likely to be predictive of how cells will react in vivo. Several presentations at SMi Group’s recent “Cell Culture” conference in London focused on advances in novel 3D cell culture systems and applications.
The things we see in 2D may not reflect what is happening in vivo, mainly due to the “lack of architecture in 2D systems, which influences the biological response in many difference phenotypes,” said Olivier Pardo, Ph.D., team leader at the Cellular Regulatory Networks Group, Imperial College, London. One clear example he cited is the response of tumor cells to drug therapy and the fact that tumor cells grown in 2D versus 3D cultures tend to respond quite differently to the same concentration of an antitumor drug. Tumor cells grown in 3D are typically more resistant to therapeutic compounds.
When cells grow in 3D spherical clusters, compared to 2D monolayers, the level of oxygenation differs depending on the whether a cell is situated more toward the inside or the outside of a spheroid structure. The difference in level of oxygenation appears to influence the response to drug therapy, explained Dr. Pardo. Additionally, differences in the extracellular matrix produced by cells growing in 3D culture systems can modify the cellular response to drugs and other stimuli, likely because of changes in integrin-based signaling in 3D compared to 2D cell cultures.
Another factor that can affect the therapeutic drug response of cells grown in culture is the extent of cell-to-cell contacts, which will tend to be more developed in 3D environments with higher cell densities.
3D culture systems are especially useful for studying the invasive properties and metastatic potential of tumor cells and for conducting screening assays for cell migration. In his presentation, Dr. Pardo described screens of large compound libraries using a bone metastatis assay or extra/intravasation assay, small-to-medium library screens with a 3D collagen invasion assay, and screening of a small number of targets using a zebrafish metastatic model organism. The 3D collagen invasion assay with confocal image acquisition was performed in 96-microwell plates.
Whereas manual screening was slow and difficult, making this assay format ill-suited for large library screening, the results obtained were highly reproducible, with >90% overlap between repeat results. In contrast, automating each step of the assay—and providing temperature control to minimize collagen polymerization—increased the throughput of the assay to make it suitable for large-scale screening, but the reproducibility of the robotized screen was poor, with <33% overlap between repeat results.
Limitations related to automation and the cost of most 3D cell culture technologies and materials available today are two important factors that stand in the way of using these methods for large-scale screening campaigns and of more widespread adoption of the technology, in Dr. Pardo’s view.
One of the main advantages of the 3D collagen assay and of 3D culture systems in general is the ability to do co-cultures, noted Dr. Pardo, thereby more closely mimicking the in vivo environment and enabling the introduction of cells capable of producing growth factors and other natural molecules to support the health and viability of the cultured cells.
An Automatable Liquid Scaffold System
At Trinity College in Dublin, Anthony Davies, Ph.D., director of the high content facility, and colleagues developed a novel liquid scaffold system that permits 3D cell culture and subsequent harvesting of the cells. The researchers designed the system to be compatible with essentially any automated liquid-handling and high-throughput screening system, as well as high-content screening and image-analysis technology.
“Unlike any other 3D assay systems currently used, our technology does not rely upon solid gel matrices, scaffolds, micropatterned surfaces, or hanging-drop assay systems to achieve reproducible cancer spheroid growth,” said Dr. Davies. “Indeed, many of the inherent technical issues surrounding these technologies are avoided using our novel 3D culture technology.”
He described the innovative liquid scaffold, the ease of doing automated 3D cell culture, and the ability to perform subsequent analyses on the isolated cultured multicellular constructs—for example, by applying biochemical, imaging, and Raman assay technologies.
This polymer-based scaffold system has the properties of a gel, yet it remains a liquid and does not solidify. The liquid has the same low viscosity, density, and properties of a regular cell culture media and is appropriate for growing virtually any cell type, according to Dr. Davies. The modified, natural polymers in the system form an elastic, reversible scaffold. Cells added to the liquid do not sink. They are able to form spherical multicellular constructs and other 3D structures. The cultured cells can be labeled, stained, exposed to experimental drugs, evaluated for their responsiveness, and essentially studied as one would cells grown in a conventional suspension culture. Addition of a deactivating agent disrupts the polymers, releasing the 3D cell structures for harvesting.
The researchers have produced this material in microliter to liter volumes. They have outlicensed the technology to Biocroi, which markets the product as Happy Cell® ASM (advanced suspension media).
Studying Drug Response in 3D Systems
The literature contains many examples of studies demonstrating differences in drug response in 3D models for monolayer systems. For instance, in a paper that appeared July 18 in the online version of Biochimica et Biophysica Acta, General Subjects, the authors (H.J. Mulhall et al.) evaluated the different electrical properties of epithelial cancer cells cultured in 2D and 3D environments and concluded that “factors such as cell shape and cytoplasmic trafficking between cells play an important role in their electrophysiology,” highlighting “the need to use in vitro models more representative of native tissue when studying cell electrophysiological properties.”
The advantages of a label-free monitoring system derived of human embryonic stem cell-derived cardiomyocyte clusters for predictive in vitro cardiotoxicity testing includes a more representative tissue milieu than traditional monolayer cell culture, as indicated in a paper that appeared July 8 in PLoS One. The authors (H.-G. Jahnke et al.) described how they monitored the adverse effects of drug exposure for more than 35 days while the clusters retained their structural and electrophysiological characteristics. The system “provides multiparameter analysis capabilities incorporating field potential recording, over days or even weeks,” said the authors.
The aim is to grow cells in 3D in vitro culture systems that mimic as closely as possible in vivo cellular microenvironments, including the effects of cell positioning within 3D constructs, cell-to-cell contacts, the extracellular matrix, cell signaling, and the factors associated with co-culture of multiple cell types. These same factors are as important in studying drug responsiveness in cultured microtissue constructs, as in evaluating the efficacy of therapeutic strategies targeting tumors.